Ning Y. Yu

SR-2508: A 2-nitroimidazole amide which should be superior to misonidazole as a radiosensitizer for clinical use☆

Abstract Using a combination of toxicological, pharmacological and radiosensitization experiments on cells in vitro and tumors in vivo , we have selected two 2-nitroimidadole amidos which should prove superior to misoaidazole (MIS) for clinical use. The two drugs, SR-2508 and SR-2555, are considered to be close to the optimum for variants of misonidazole of the same electron affinity; their main difference is a lower lipophilicity than MIS. This lower lipophilicity leads to poorer penetration into neural tissues than that of MIS and this leads, as expected, to lower neurotoxicity of these drugs compared to MIS (as judged by an accelerating rotarod test following 4–5 weeks of daily injections). The in vitro radiosensitization tests on hypoxic Chinese hamster ovary HA-1 cells show that SR-2508 and MIS have the same radiosensitization efficiency but are slightly more potent as radiosensitizers than SR-2555. For the in vivo radiosensitization experiments we used the in vivo-in vitro cloning assay with the EMT6 tumor, the regrowth assay with RIF-1 and KHJJ tumors and the TCD 50 assay with the MDAH/MCa4 carcinoma. All show roughly equivalent levels of radiosensitization of both SR-2508 and SR-2555 to MIS although, as predicted by the in vitro results, SR-2555 is slightly inferior to SR-21.508. We conclude that, although SR-2555 appears to be slightly less neurotoxic than SR-2508, its lower radiosensitizing efficacy is likely to make it less attractive than SR-2508 as a replacement for MIS for clinical use. Extrapolating the neurotoxicity data obtained in the mouse together with pharmacological studies of SR-2508 in spontaneous tumors of the dog, we estimate that levels of SR-2508 of at least 7.5 times those of MIS will be able to be achieved in human tumors for the equivalent level of neurotoxicity. This should enable essentially maximum radlosensitization of the hypoxic cells in human tumors to be obtained in conventional daily fractionation.

The use of drugs which deplete intracellular glutathione in hypoxic cell radiosensitization

Diethylmaleate (DEM) is a thiol-binding reagent with specificity toward glutathione. Treatment of Chinese hamster ovary (CHO) cells in vitro with 2 x 10(-4) M DEM for one hour results in a decrease in glutathione content to less than 5% of control, without cytotoxicity. This treatment results in dose-modifying sensitization to radiation under hypoxic conditions, with no effect on the shoulder of the radiation survival curve. No effect on the radiation sensitivity of oxygenated cells was seen. DEM pretreatment enhances the radiosensitization of hypoxic cells by misonidazole, as well. Similar results were obtained in vivo with EMT6 tumors in BALB/c mice. Analysis of DNA damage by the alkaline elution assay indicates that DEM enhances radiation-induced single-strand breaks, but does not significantly affect repair, while diamide and N-ethylmaleimide inhibit repair, in addition to enhancing radiation-induced single-strand breaks.

Intratumoral radioimmunotherapy of a human colon cancer xenograft using a sustained-release gel

Low tumor uptake and normal tissue toxicity limit the efficacy of RIT for the treatment of solid tumors. In this study, an intratumoral injectable gel drug delivery system for local administration of RIT was evaluated using the LS174T human colon cancer xenograft model in SCID mice. The injectable gel is a collagen-based drug delivery system designed for intratumoral (i.t.) administration, which has previously been shown to enhance drug retention at the injection site and reduce systemic drug exposure. We compared the local (tumor) retention and biodistribution of 111In-labeled NR-LU-10 monoclonal antibody given i.t. in the injectable gel versus simple aqueous solution. 111In gel given i.t. and 111In-NR-LU-10 given intraperitoneally (i.p.) were used as controls. The results showed that tumors treated with 111In-NR-LU-10 gel maintained the highest levels of radioactivity for up to 96 h. At 48 h after the administration of 111In-NR-LU-10 gel i.t., 111In-NR-LU-10 solution i.t., 111In gel i.t., or 111In-NR-LU-10 i.p., the level of radioactivity remaining in each gram of tumor was 98, 49, 45, and 16% of the injected dose, respectively. It was estimated that if 100 microCi of 90Y-NR-LU-10 were administered similarly, tumor treated with 90Y-NR-LU-10 gel i.t. would receive a dose of 90.0 Gy, whereas normal tissues in the same animal would receive a dose of approximately 2.43 Gy. In contrast, if 90Y-NR-LU-10 were delivered i.p., a comparable tumor would receive a dose of 16.8 Gy and corresponding normal tissues would receive 3.36 Gy. Consistent with these estimates, enhanced antitumor efficacy was observed when 90Y-NR-LU-10 gel was administered i.t. Tumor growth delay time was 6.9-fold (P < 0.01) longer in these animals (14.4 days) than in animals treated with 90Y-NR-LU-10 i.p. (2.1 days). Systemic toxicity was also significantly reduced in gel-treated animals as monitored by loss of body weight. This study demonstrated that intratumoral delivery of 90Y-NR-LU-10 gel markedly increased the retention of the radioisotope in tumors, enhanced the antitumor efficacy, and reduced systemic toxicity compared to systemic administration of the radiolabeled antibody. This injectable gel drug delivery system may allow for improvement in the therapeutic index for RIT.

Depletion of glutathione in vivo as a method of improving the therapeutic ratio of misonidazole and SR 2508

Depletion of intracellular glutathione (GSH) can enhance misonidazole (MISO) radiosensitizing efficacy both in vivo and in vitro. However, such treatments may also enhance the systemic toxicity in animals. The purpose of the present study was to test various ways of depleting GSH levels in a variety of experimental mouse tumors, to measure the improvement in the efficacy of MISO and its less toxic analog SR 2508 by this depletion, and to determine the effect of daily GSH depletion on the toxicity of MISO and SR 2508. GSH levels were measured daily for 5 days in tumors, livers and brains of mice injected daily with buthionine sulfoximine (BSO), with or without diethylmaleate (DEM). To investigate tumor variability we studied 5 different tumors: EMT-6, RIF-1, KHT, SCC VII, and B16 melanoma. The efficacy of MISO and SR 2508 was evaluated using the KHT and SCC VII tumors either by the regrowth delay assay or by the in vivo/in vitro clonogenic assay. The drug toxicity was evaluated by weight loss and by death. Daily doses of 3 mmole/kg BSO depleted tumor levels of GSH to 20 to 40% of controls by 6 hr after each injection. Injection of DEM (300 mg/kg) 6 hr after BSO further enhanced the depletion. Administration of MISO or SR 2508 at the time of maximum GSH depletion enhanced the MISO efficacy by factors of 2.5 to 8 for depletion to 8% of controls by BSO + DEM, but no enhancement of SR 2508 was seen with tumors at 20% GSH levels achieved with BSO alone in the preliminary experiment. The chronic toxicity of MISO was enhanced not at all or by a factor of up to 2 for BSO and BSO + DEM respectively. Further studies are needed before it can be concluded that GSH depletion by BSO alone may be a useful adjunct to the clinical use of radiosensitizers.

Drug retention and distribution after intratumoral chemotherapy with fluorouracil/epinephrine injectable gel in human pancreatic cancer xenografts

Purpose: Pancreatic cancer is widespread, associated with high mortality, and rapidly fatal. Most cases are diagnosed too late for surgical treatment, and the disease responds poorly to systemic chemotherapy. Nevertheless, pancreatic cancer cells are sensitive to fluorouracil (5-FU) in a time- and dose-dependent manner, suggesting that improved retention of drug in the tumor may improve patient prognosis. In this study, we evaluated a novel drug delivery system, 5-FU/epinephrine injectable gel (5-FU/epi gel), designed to improve drug retention in tumors. Methods: We used a BxPC-3 human pancreatic cancer xenograft model in athymic mice to examine drug levels in tumor, liver, and kidney tissue following administration of: (a) 5-FU/epi gel (30 mg 5-FU/ml) intratumorally (i.t.); (b) 5-FU solution i.t.; and (c) 5-FU solution intraperitoneally (i.p.). [3H]5-FU was added as a radiolabeled marker to all test formulations. Animals were sacrificed at designated times, and the tumor, liver, and one kidney from each animal were excised and processed for radioactivity analysis. Drug concentration was quantified by both storage-phosphor autoradiography (SPA) and liquid scintillation counting (LSC). Results: Higher and sustained i.t. drug levels were achieved following i.t. administration of 5-FU/epi gel (SPA AUC 18.4 mM · h, LSC AUC 13.0 mM · h) compared with 5-FU solution i.t. (SPA AUC 2.02 mM · h, LSC AUC 1.92 mM · h) or 5-FU solution i.p. (SPA AUC 0.07 mM · h, LSC AUC 0.04 mM · h). Use of the 5-FU/gel system was associated with lower drug levels in liver and kidney, indicating that it produces far less systemic exposure. Conclusion: In the human pancreatic cancer xenografts, i.t. administration of 5-FU/epi injectable gel provided significantly higher drug and/or metabolite concentrations for extended periods than was possible with either i.t. or i.p administration of drug solution. This i.t. drug delivery system could potentially be used to treat patients with pancreatic cancer to increase tumor exposure to drug and improve the therapeutic index in comparison to systemic drug administration.

Radiosensitization of hypoxic cells in vivo by SR 2508 at low radiation doses: a preliminary report.

We have used the RIF-1 tumor implanted intradermally in the lower dorsum of C3H mice to explore to what extent the radiosensitizer SR 2508 is capable of sensitizing hypoxic cells at clinically relevant doses of 1 and 2 Gy per fraction. We injected SR 2508 (1000 mg/kg) 45 min prior to each radiation dose in fractionated regimens of 2 or 4 doses/day for up to 5 days (1 or 2 Gy/fraction) given locally to the tumors, which were clamped to occlude the blood supply prior to each radiation exposure. This necessitated the design of clamps which totally occluded blood flow could be applied to nonanesthetized mice without obvious discomfort, and could be applied up to 20 times without compromising the tumor blood supply on removal of the clamps. We have performed various experiments which confirm the validity of these 3 requirements. The response of the tumor cells with and without clamping and with and without SR 2508 was determined by constructing multifraction cell survival curves using the in vivo-in vitro assay. The initial results demonstrate significant radiosensitization of artificially hypoxic tumor cells at 1 and 2 Gy/fraction by SR 2508 (1000 mg/kg). Using the ratio of the D0s of the exponential, multifraction survival curves, we obtained an SER for SR 2508 of 1.6 (3 experiments pooled) compared to an OER (D0 clamped/D0 air-breathing mice) of 2.3 (4 experiments pooled). These data suggest that SR 2508 (and presumably other electron-affinic sensitizers) can radiosensitize hypoxic cells at low radiation doses, and indicate that this and similar drugs may be useful in the radiotherapy of those tumors for which hypoxia limits curability.

Intratumoral chemotherapy with a sustained-release drug delivery system inhibits growth of human pancreatic cancer xenografts.

This study provides the first evidence that treatment of human pancreatic adenocarcinoma is markedly improved by the intratumoral administration of chemotherapeutic agents in a novel drug delivery system. The effect of chemotherapeutic agents delivered in a sustained-release, protein-based, injectable gel was evaluated on the growth of human pancreatic adenocarcinoma cell line, BxPC-3. In vitro chemosensitivity of BxPC-3 cells exposed for 24 or 72 h to fluorouracil (0.01-5 mM), cisplatin or doxorubicin (0.1-50 microM) and floxuridine, vinblastine, mitomycin or paclitaxel (1.0-100 microM) was compared with that of untreated cells. In vitro chemosensitivity was also studied with fluorouracil and mitomycin in the poorly differentiated PANC-1, human pancreatic cancer cell line. Survival was determined after 7-10 days. All drugs decreased cell growth in a dose-dependent fashion. The efficacy of fluorouracil, cisplatin and doxorubicin increased with prolonged exposure, rendering these drugs most appropriate for a sustained-release preparation. For in vivo studies, athymic nude mice bearing BxPC-3 xenografts were treated either with fluorouracil, cisplatin or doxorubicin in the therapeutic injectable gel containing epinephrine or with vehicle alone administered intratumorally on days 1 and 4. After 28 days, the mice were sacrificed and tumors dissected and weighed. Tumors in mice treated with the injectable gel decreased in size by 72-79% compared with tumors in untreated controls and tumors treated with vehicle alone. Intratumoral injection of drug solution and intraperitoneal injection of drug in the injectable gel did not change tumor size compared with controls. In a drug-retention study, mice were injected intratumorally with [3H]fluorouracil either in the injectable gel or in solution. Sustained radioactivity was observed in tumors injected with the gel, and, conversely, greater radioactivity was detected in the liver and kidneys in mice receiving the radiolabeled solution. These results suggest that the therapeutic injectable gel chemotherapy, when given intratumorally, may improve tumor response with less systemic toxicity in comparison with conventional systemic chemotherapy.

Antitumor effect of intratumoral administration of fluorouracil/epinephrine injectable gel in C3H mice

Fluorouracil/epinephrine injectable gel (5-FU/epi gel) was evaluated in vitro for its drug-release profile characteristics and in a mouse tumor model for its antitumor effectiveness. In vitro chemosensitivity studies with 5-FU in RIF-1 fibrosarcoma cells showed less than 1 log cell kill at 1 mM after 2 h of exposure. Increasing the exposure time to 24 h resulted in greater cell killing (∼ 2.5 log cell kill at 0.5 mM), suggesting that sustained drug levels in tumors would result in an increased efficacy outcome in vivo. A 5-FU/epi injectable gel was designed, providing drug release in vitro of 50% by ∼ 4 h and of 80% by 24 h. The retention of 5-FU in RIF-1 mouse tumors was determined after intratumoral administration of 5-FU/epi gel or various combinations of the formulation components. Area-under-the-curve (AUC0–24 h) calculations resulted in an AUC value of 146.4% h for the 5-FU/epi gel formulation as compared with 45.7% h for 5-FU solution. Tumor growth was significantly delayed (P<0.05) with the 5=FU/epi gel (60 mg/kg) as compared with 5-FU solution given intratumorally or systemically. A fluorouracil dose of 150 mg/kg in the 5-FU/epi gel given weekly for 13 weeks was not lethally toxic, whereas the same dose given as drug solution was 100% lethal, suggesting that the therapeutic index for 5-FU in the gel formulation may be much greater than that for aqueous drug solution delivered intratumorally.

In vitro and in vivo radiosensitization by 2-nitroimidazoles more electron-affinic than misonidazole

A series of 5-substituted-methyl-2-nitroimidazoles, more electron-affinic than misonidazole (MISO), has been studied as potential hypoxic cell radiosensitizers. In vitro radiosensitization studies of these compounds showed equivalent or greater radiosensitization than MISO, while LD50 studies of the compounds found them to be, in general, more toxic to Balb/c mice than MISO. Radiosensitization experiments in vivo with compounds SR-2537, SR-2515 and SR-2553 of acceptable toxicity were not able to sensitize the EMT6 tumor to x-irradiation after a single intraperitoneal injection. However, moderate sensitization was achieved when SR-2537 was administered i.v. Rapid metabolism of these more electron-affinic compounds was suggested as a possible cause of the poor sensitization. However, when multiple i.v. injections of SR-2537 were given to maintain a constant drug level in the tumor, radiosensitization by this compound did not improve, suggesting that intact drug was either not reaching or was not penetrating the hypoxic cells.